Project Summary/Abstract Chagas disease (CD), caused by infection with T. cruzi parasites, is a major but under-studied cause of heart failure in the Americas. Over seven million people are T. cruzi-positive, including at least 300,000 in the USA. T. cruzi-infected individuals initially progress through an acute disease stage with high parasite burden. Immune control of parasites leads to transition to an asymptomatic disease stage. 30-40% of infected individuals will then progressively develop severe cardiac and/or digestive symptoms (symptomatic chronic CD), with an annual mortality rate of over 12,000/year. No vaccines are available, and current treatment options are limited to two drugs, benznidazole (Bz) and nifurtimox. These antiparasitic agents, although able to effectively kill T. cruzi, have significant side effects and are unable to improve cardiac function in advanced chronic CD. There is therefore an urgent need for new treatments for CD. Combination therapy development has been identified as a major CD research priority by the WHO. We have previously demonstrated that levels of acylcarnitine molecules are altered in severe vs mild CD. We have also generated pilot data demonstrating that L-carnitine (LC) treatment in acute-stage CD prevents animal mortality, and that LC treatment in a chronic-stage CD mouse model reduces indicators of heart dysfunction. Strikingly, these effects occurred without changes in parasite burden, supporting a mechanism by which LC treatment reduces CD-mediated cardiac damage. This would make LC an ideal adjunct to existing antiparasitic regimens that do not improve heart function in late-stage CD. We therefore hypothesize that LC+Bz combination regimens are safe and effective to treat chronic-stage CD, with Bz killing T. cruzi and LC improving heart function. This exploratory/developmental R21 project will focus on rigorously assessing the potential of this combination regimen for chronic CD treatment, as a necessary precursor for further pre-clinical and clinical development of LC +Bz combination regimens. In aim 1, we will assess the efficacy of different LC+Bz combination regimens in terms of parasite clearance and reduction in cardiac damage, when administered in the chronic stage of CD. In aim 2, to meet FDA guidelines for combinations of approved drugs in which one combination member has significant toxicity (Bz) and there is pharmacokinetic interaction potential, we will assess overall safety and tolerability of different LC+Bz combination regimens. These complementary efficacy and safety results will determine which LC+Bz combinations should progress for further evaluation through the pre-clinical drug development pipeline and ultimately to human clinical trials for chronic CD. Importantly, LC is cheap, readily available as a dietary supplement, and FDA-approved to treat metabolic disorders. This proposal therefore has great potential for rapid bench-to-bedside translation and improvement of CD patient outcomes.